Proper regulation of cell division is required for normal growth, development and genomic integrity; aspects of this control are lost or disrupted in human cancers. The cyclin-dependent kinases (CDKs) drive all major transitions in the cell division cycle. Mammalian cells have multiple downstream effector CDKs but, as we show, a single upstream CDK-activating kinase (CAK), Cdk7. Gene knockout and silencing studies suggested redundancy in the CDK network, by showing that Cdk2-long thought to be the principal driver of DNA synthesis (S) phase-was dispensable for viability. However, our work-using a chemical-genetic approach that selectively inactivates catalytic function of the targeted CDK while sparing non-catalytic, scaffold functions-uncovered strict requirements for Cdk2 activity in proliferation of both transformed and non- transformed human cells. An important function of Cdk2 is to prevent premature activation of Cdk1, and thereby ensure coordinated progression through S phase. That function depends on a kinetically more favorable pathway for activation of Cdk2, compared to Cdk1, due in part to different mechanisms of recognition by Cdk7. I hypothesize that specialized functions of Cdk2-in regulating gene expression, coordinating S phase and responding to DNA damage or replication stress-emerge from its unique mode of activation. I propose, moreover, that another distinct pathway by which Cdk7 activates Cdk4 and Cdk6-which function prior to cell-cycle commitment-is directly or indirectly coupled to mitogen-sensing pathways. The specific aims are: 1. To dissect the G1/S regulatory network comprising Cdk7, Cdk2, Cdk4 and Cdk6 2. To probe specific functions of Cdk7 and Cdk2 in the DNA damage response 3. To target the Cdk2 activation pathway with small molecule inhibitors Our preliminary studies reveal a unitary CAK-CDK network that achieves regulatory flexibility through kinetically distinct modes of kinase activation and inactivation. I ow propose to probe specific functions of CAK and the downstream CDKs in coordinating constitutive cell-cycle events and ensuring effective responses to genotoxic insults; and to test a new paradigm for inhibiting a specific CDK by selective targeting of its activation pathway. These studies promise to advance basic understanding of cell-cycle control, and to reveal novel strategies for anti-CDK therapy of human cancer.